Glucagon is a 29 amino acid peptide hormone produced in the pancreatic islets of Langerhans. Multiple forms of immunoreactive glucagon-like peptides of molecular weights from 3,500 to 200,000 have been identified in islets intestine and brain. This indicates that glucagon is synthesized as a large molecular weight precursor. Recently, we identified two precursors to glucagon, pre-proglucagons, of apparent Mr 14,500 and 12,500 among products of cell-free translation of mRNAs from anglerfish islets. We prepared complementary DNAs (dscDNAs) to the islet pre-proglucagon mRNAs and cloned these into bacterial hosts. Nucleotide sequence analyses of dscDNAs encoding the two pre-proglucagons demonstrated that the two different pre-proglucagons are the products of different mRNAs and different genes. Amino acid sequences of the two precursors derived from the sequences of dscDNAs demonstrated that each of the precursors contains two glucagon-related sequences arranged in tandem. In preliminary studies we identified mRNA in extracts of fish intestine which hybridize to islet dscDNAs encoding glucagon, and a rat glucagon precursor of Mr 20,000 was identified among products of cell-free translation of mRNA prepared from rat neonatal pancrease. We propose to (1) characterize mRNAs encoding glucagon in fish and rat islets and intestine using fish islet glucagon cDNAs as probes (2) select and perform nucleotide sequence analyses on glucagon related dscDNAs from cloned dscDNA libraries of fish intestine, and rat neonatal pancreas and foetal intestine. (3) select genomic DNA sequences from lambda phage libraries of rat genomic DNA, and to determine the structures of rat glucagon genes. (4) to investigate co-translational processing of glucagon precursors by isolation and microsequencing of labeled proglucagons. (5) to investigate post-translational processing of glucagon precursors by in situ immunohistochemical studies with antibodies to synthetic peptide fragments of proglucagons. (6) to investigate the ontogenetic regulation of glucagon mRNA production in foetal and neonatal rat pancreas by using rat glucagon dscDNAs in hybridization assays. The proposed studies will provide information about the structures of glucagon-related precursors and the genes encoding them and to tools to investigate the regulation of glucagon gene expression.